The present invention is generally concerned with biosensors, biosensor arrays, and sensing apparatus, and sensing methods for the analysis of chemical and biological materials. More particularly, the invention is directed to biosensors, biosensor arrays, sensing apparatus and sensing methods which employ cells and mixed populations of cells for analysis of chemical and biological materials.
It is generally recognized that important technical advances in chemistry, biology and medicine benefit from the ability to perform microanalysis of samples in minute quantities. However, making analytical measurements on minute quantities has long been a challenge due to difficulties encountered with small volume sample handling, isolation of analytes, and micro-analysis of single-cell physiology.
Nanoliter, picoliter, and femtoliter volume studies have been explored in a range of applications involving in vitro and in vivo cellular investigations [R. M. Wightman, et al., Proc. Natl. Acad. Sci. U.S.A. 88:10754(1991); R. H. Chow, et al. Nature 356:60(1992); T. K. Chen, et al. Anal. Chem. 66:3031(1994); S. E. Zerby, et al., Neurochem. 66:651(1996); P. A. Garis, et al. J.Neurosci. 14:6084(1994); G. Chen, et al., J.Neurosci. 15:7747(1995)], electrochemistry [R. A. Clark, et al., Anal. Chem. 69(2):259(1997)], matrix-assisted laser desorption-ionization mass spectrometry [S. Jespersen, et al., Rapid Commun. Mass Spectrom. 8:581(1994)], micro-column liquid chromatography [I. A. Holland, et al., Anal Chem. 67:3275(1995); M. D. Oates, et al., Anal. Chem. 62:1573(1990)], micro-titration [M. Gratzl, et al Anal. Chem. 65:2085(1993); C. Yi, et al., Anal. Chem. 66:1976(1994)], and capillary electrophoresis [M. Jansson, et al., J.Chromatogr. 626:310(1992); P. Beyer Hietpas, et al. J.Liq.Chromatogr. 18:3557(1995)].
Clark, et al. [Anal. Chem. 69(2):259(1997)] has disclosed a method for fabricating picoliter microvials for electrochemical microanalysis using conventional photolithographic masking and photoresist techniques to transfer mold polystyrene microvials on silicon wafer templates. These microvials typically exhibit non-uniformity in size and shape due to the difficulty in controlling the resist etching of the molding surface and the transfer molding process.
Park, et al. [Science 276:1401(1997)] has disclosed a modified lithographic method for producing arrays of nanometer-sized holes using polystyrene-polybutadiene, ordered, diblock copolymers as masks in reactive ion etching of silicon nitride. This multi-step method is capable of producing arrays of picoliter-sized holes which are typically 20 nanometers in diameter and 20 nanometers deep with a spacing of 40 nanometers. Hole densities of up to 1011 holes/cm2 are disclosed. The range of sizes and spacings of the holes produced by this method is limited by the size of the copolymer microdomains. Uniformity of hole size and spacing is difficult to maintain with this method due to difficulties in controlling the etching method employed to form the holes.
Deutsch, et al. [Cytometry 16:214(1994)] have disclosed a porous electroplated nickel microarray comprised of micron-sized conical holes in blackened nickel plate. Hole sizes range from a 7 um upper diameter to a 3 um lower diameter with an 8 um depth. The array is used as a cell carrier for trapping individual cells while studying the responses of individual cells to changes in their microenvironment. In U.S. Pat. No. 4,772540, Deutsch, et al., have also disclosed a method for making such an array using a combined photoresist and electroplating technique.
Coming Costar Corp. (Acton, Ma) produces a commercial microwell array for miniaturized assays under the trademark PixWell(trademark). These arrays are made from microformed glass plates and comprise 40 um diameter by 20 um deep tapered wells with a well density of 4356 wells/cm2.
Microwell arrays have particular utility in the study of living cells. In cell research, the measurement of responses of individual cells to changes or manipulations in their local environment is desirable. Any method or device designed for such studies must provide for the capability of maintaining cell viability, identifying the location of individual cells, and correlating response measurements with individual cells.
Due to the availability of viable fluorescent probes for intracellular studies, fluorescence measurements of living cells have significant utility in the study of cell functions. Thus fluorescence optical measurements are often utilized in cell studies where three generic methods of cell measurement are available, comprising bulk measurements of cell populations, dynamic measurements of cell populations or individual cells, and static measurements of individual cells.
The characteristics of an entire cell population as a whole can be studied with bulk measurements of sample volumes having a plurality of cells. This method is preferred where cell populations are very homogeneous. A generally recognized limitation of this method is the presence of background fluorescence which reduces the sensitivity of measurements and the inability of distinguishing differences or heterogeneity within a cell population.
Flow cytometry methods are often employed to reduce problems with background fluorescence which are encountered in bulk cell population measurements [M. R. Gauci, et al., Cytometry 25:388(1996); R. C. Boltz, et al., Cytometry 17:128(1994)]. In these methods, cell fluorescence emission is measured as cells are transported through an excitation light beam by a laminar flowing fluid. Flow cytometry methods may be combined with static methods for preliminary sorting and depositing of a small number of cells on a substrate for subsequent static cell measurements [U.S. Pat. No. 4,009,435 to Hogg, et al.; Kanz, et al., Cytometry 7:491(1986); Schildkraut, et al., J.Histochem Cytochem 27;289(1979)].
Gauci, et al., disclose a method where cell size, shape and volume is measured by light scattering and fluorescent dyes are utilized to determine protein content and total nucleic acid content of cells. This method further provides for counting and sizing various cells at a rate of approximately 100 cells per second.
Flow cytometry techniques are generally limited to short duration, single measurements of individual cells. Repetitive measurements on the same cell over time are not possible with this method since typical dwell times of a cell in the excitation light beam are typically a few microseconds. In addition, the low cumulative intensity from individual cell fluorescence emissions during such short measurement times reduces the precision and limits the reliability of such measurements.
Regnier, et al., [Trends in Anal.Chem. 14(4):177(1995)] discloses an invasive, electrophoretically mediated, microanalysis method for single cell analysis. The method utilizes a tapered microinjector at the injection end of a capillary electrophoresis column to pierce an individual cell membrane and withdraw a sample of cytoplasm. The method measures cell contents, one cell at a time. The method is generally limited to the detection of easily oxidized species.
Hogan, et al., [Trends in Anal.Chem. 12(l):4(1993)] discloses a microcolumn separation technique which may be utilized in combination with either a conventional gas chromatograph-mass spectrometer, micro thin layer chromatography or high pressure liquid manipulation of small cellular volumes. The sensitivity of the method is limited and may require pre-selection of target compounds for detection.
Static methods are generally the preferred method for measurements on individual cells. Measurement methods range from observing individual cells with a conventional optical microscope to employing laser scanning microscopes with computerized image analysis systems [see L. Hart, et al., Anal. Quant. Cytol. Histol. 12:127(1990)]. Such methods typically require the attachment of individual cells to a substrate prior to actual measurements. Problems are typically encountered in attaching single cells or single layers of cells to substrates and in maintaining cells in a fixed location during analysis or manipulation of the cell microenvironment. Additionally, repetitive measurements on individual cells typically require physically indexing the location of individual cells and providing a mechanism for scanning each cell sequentially and returning to indexed cell locations for repeated analysis of individual cells.
Huang, et al., [Trends in Anal. Chem., 14(4)158(1995)] discloses a static electrochemical method and electrode for monitoring the biochemical environment of single cells. The method requires fabrication and manual positioning of a microelectrode reference and working electrode within the cell. The method has been used to detect insulin, nitric oxide and glucose inside single cells or external to the cells. The method is generally limited to the study of redox reactions within cells.
Ince, et al. [J.Immunol. Methods 128:227(1990)] disclose a closed chamber device for the study of single cells under controlled environments. This method employs a micro-perfusion chamber which is capable of creating extreme environmental conditions for cell studies. Individual cells are held in place by two glass coverslips as various solutions are passed through the chamber. One limitation of the method is the difficulty in eliminating entrapped gas bubbles which cause a high degree of autofluorescence and thus reduces the sensitivity of measurements due to background fluorescence.
In an attempt to overcome the limitations encountered with conventional static methods, Deutsch, et al., [Cytometry 16:214(1994)] and Weinreb and Deutsch, in U.S. Pat. Nos. 4,729,949, 5,272,081, 5,310,674, and 5,506,141, have disclosed an apparatus and method for repetitive optical measurements of individual cells within a cell population where the location of each cell is preserved during manipulation of the cell microenvironment.
A central feature of the apparatus disclosed by Deutsch, et al., is a cell carrier, comprising a two dimensional array of apertures or traps which are conical-shaped in order to trap and hold individual cells by applying suction. The cell carrier is typically fabricated by the combined electroplating-photoresist method disclosed in U.S. Pat. No. 4,772540 to Deutsch, et al. The purpose of the cell carrier is to provide a means for maintaining the cells in fixed array locations while manipulating the cell environment. Individual cells are urged into cell carrier holes by suction and the wells are subsequently illuminated with a low intensity beam of polarized light that reads back-emitted polarization and intensity. Measurements are compared when two different reagents are sequentially reacted with the cells. The method as disclosed requires two separate cell carriers for both a baseline control and analyte measurement.
The method and device of Deutsch, et al., have been employed by pathologists in diagnostic tests to determine the health and viability of cell samples taken from patients. The method and device have been applied to both cancer screening [Deutsch, et al., Cytometry 16:214(1994), Cytometry 23:159(1996), and European J.Cancer 32A(10):1758(1996)] and rheumatoid arthritis [Zurgil, et al., Isr.J.Med.Sci. 33:273(1997)] in which fluorescence polarization measurements are used to differentiate lymphocytes of malignant versus healthy cells based on changes in the internal viscosity and structuredness of the cytoplasmic matrix induced by exposure to tumor antigen and mitogens.
The method and device disclosed by Deutsch, et al., requires employment of a scanning table driven by three stepping motors and a computer control system for mapping, indexing and locating individual cells in the cell carrier. The use of such mechanical scanning methods introduces limitations in reproducibility and accuracy of measurements due to conventional mechanical problems encountered with backlash and reproducible positioning of individual cell locations for repeated measurements. In addition, mechanical scanning of the entire array prolongs the measurement time for each cell in the array.
The method disclosed by Deutsch, et al., is further limited by the use of fluorescence polarization measurements which have certain intrinsic limitations due to the significant influence of various optical system components on polarization as the fluorescence emission response is passed from the cell carrier to optical detectors. Birindelli, et al. [European J. Cancer 33(8):1333(1997)], has also identified limitations in this method due to fluctuations in electropolarisation values which require taking averages of at least three measurement scans for each condition so as to obtain reliable measurements. In addition, for cell studies, polarization measurements are generally limited to cell responses which produce sufficient changes in cytoplasm viscosity to produce a detectable change in polarization. Since not all cell responses are accompanied by detectable viscosity changes, the method is further limited to the cell activities which create such viscosity changes in the cytoplasm.
Zare, et al., [Science 267:74(1995); Biophotonics International, March-April, pl7 (1995)] discloses a biosensor system based on the response of living cells to complex biological materials fractionated by a microcolumn separation technique. Cells which were positioned on a glass cover slip were treated with a fluorescent probe and subsequently shown to be sensitive to a series of biological compounds including acetylcholine, bradykinin, and adenosine triphosphate as well as changes in intracellular calcium levels.
Yeung, et al. [Acc. Chem. Res. 27:409(1994)] has reviewed a number of methods for single cell response studies and has observed a significant variation and heterogeneity within cell populations based on analyte measurements. For example, the reference discloses a capillary electrophoresis method for exposing cells to biologically reactive compounds, extracting the intracellular fluid of individual cells produced in response to such compounds, and identifying analytes from migration times in the capillary column. Other fluorescence-based assays are also disclosed. Significant cell-to-cell variations and heterogeneity in individual cell responses within a cell population were observed which differences could provide a means for discriminating between biological and chemical compounds in contact with individual cells.
McConnell, et al. [Science, 257:1906(1992)], disclose a microphysiometer device known as the xe2x80x9cCytosensorxe2x80x9d which uses a light addressable potentiometer sensor to measure the rate at which cells acidify their environment. This sensor acts as miniaturized pH electrode for monitoring cell responses which produce detectable changes in local pH. The disclosed device is limited to the measurement of proton excretions from cells and thus is only capable of detecting acidic cell responses to analytes.
U.S. Pat. No. 5,177,012 to Kim, et al., disclose a biosensor for the determination of glucose and fructose. The biosensor is produced by treating whole cells with an organic solvent and immobilizing the treated cells residue on a support to form a whole cell membrane which is applied to a pH electrode.
U.S. Pat. No. 5,690,894 to Pinkel, et al., discloses a biosensor which employs biological xe2x80x9cbinding partnersxe2x80x9d, materials such as nucleic acids, antibodies, proteins, lectins and other materials derived from cells, tissues, natural or genetically-engineered organisms. These agents are used in conjunction with a fiber optic array where each species of binding partners is uniquely addressed by a group of fibers within the fiber optic bundle which is coupled to an optical detector. The array was designed for screening of extensive arrays of biological binding partners.
While many of the prior art methods provide for the analysis of either single cells or populations of cells and some of these methods provide for monitoring cell responses to target analytes, none of the disclosed methods provides for employing large populations of monocultures or mixed populations of living cells for simultaneously monitoring the responses of individual cells to biological stimuli produced by chemical and biological analytes. Thus there is a need for a biosensor array and method which efficiently utilizes the ability of populations of living cells to respond to biologically significant compounds in a unique and detectable manner. Since the selectivity of living cells for such compounds has considerable value and utility in drug screening and analysis of complex biological fluids, a biosensor which makes use of the unique characteristics of living cell populations would offer distinct advantages in high throughput screening of combinatorial libraries where hundreds of thousands of candidate pharmaceutical compounds must be evaluated. In addition, such a sensor would be useful in monitoring bioprocesses and environmental pollution where the enhanced sensitivity of living cells to their environment can be exploited.
In general, the invention provides for a biosensor, a biosensor array, a biosensor sensing system and sensing methods for the analysis of chemical and biological materials. More particularly, the invention provides for biosensors and biosensor arrays, sensing apparatus and sensing methods which employ living cells and mixed populations of living cells for analysis of chemical and biological materials.
The biosensor array of the present invention comprises either a monoculture of living cells or randomly mixed populations of living cells wherein each individual cell in the array is positioned in an optically addressable microwell which is preformed to accommodate the size and shape of the individual cells. The biosensor array sensing method relies on the well known fact that individual cells, which are chemically or biologically stimulated by the presence of a biological or chemical material in the cell environment, will respond by producing a change in the cell or cellular environment which can be optically interrogated and detected within the cell itself or from an indicator compound, for example, a fluorophore, chromophore or dye, either attached to the cell, taken up in the cell, or added to the local cell environment. The biosensor of the present invention thus capitalizes on the ability of living cells to respond to biologically significant compounds. Since the selectivity of living cells for such compounds has considerable value and utility in drug screening and analysis of complex biological fluids, the biosensor of the present invention offers distinct advantages to high throughput screening of combinatorial libraries where hundreds of thousands of candidate compounds must be evaluated.
In a preferred embodiment, the biosensor array of the present invention is incorporated into a fiber optic array. In this embodiment, the distal end of each fiber strand in a fiber optic bundle or fiber optic array is chemically etched so as to create a cavity or microwell. A schematic diagram of the biosensor array concept of the present invention is shown in FIG. 1. In a preferred embodiment, individual living cells of either a monoculture of cells or mixed populations of cell lines are deployed in the microwells. The microwells are formed by anisotropic etching of the cores of the individual fiber in the fiber bundle or fiber array. The microwells are formed by controlling the etching process so as to remove a centralized core portion of the individual fiber strands while leaving the surrounding cladding intact. The resultant etched cavity is dimensioned for accommodating an individual cell. By selecting a fiber optic bundle or fiber optic array whose individual fiber cores are appropriately sized and by careful control of the etching conditions, the diameter and depth of the microwells can be controlled and adjusted over any convenient dimension range so as to match the size of any desired cell type.
In one embodiment, the interior surfaces of the microwells may be coated with a thin film of biologically inert material such as collagen, fibronectin, polylysine, polyethylene glycol, polystyrene, or a metal such as gold, platinum or palladium. In an alternative embodiment, an indicator compound, for example, a fluorophore, a chromophore or dye, may be attached to the microwell surface for detecting cell responses to chemical or biological stimulation.
By incorporating a biosensor into a fiber optic array, the innovation of the biosensor of the present invention is in providing for optical coupling of individual cells located in microwells with discrete individual optical fibers in a fiber optic array or bundle. Since typical fiber optic arrays contain thousands of discrete individual fiber strands, the invention thus provides for the individual optical coupling and interrogation of thousands of cells within an array, thereby providing for a large number of independent cell response measurements for each cell population within an array. Due to both the number of cell populations available and the correspondingly large number of individual cells within each cell population, a significant innovation of the present invention is in providing for the summing and amplification of the characteristic optical response signatures of multiple independent measurements taken from cells within each cell population, thereby improving the detection limit and sensitivity of the biosensor.
An additional innovation of the present invention is that, by deploying a large number of cell populations within the array, and providing a large number of individual cells in each population, the discriminating capabilities of the biosensor array toward biological or chemical analytes is significantly enhanced by providing for thousands of cell responses from a large number of cell populations. This feature directly mimics the actual behavior of the human olfactory system where the combined signals from thousands of receptor cells, in each grouping of nearly a thousand different receptor cell types found in the epithelium layer, none of which are particularly sensitive in themselves, lead to a highly amplified sensory response to odors [see Kauer, et al, Trends Neurosci. 14:79(1991). One embodiment of the present invention thus mimics the evolutionary scent amplification process found in the human olfactory system in order to significantly enhance biosensor array sensitivity to analytes by summing the low-level responses of a large number of cells in the biosensor array. By summing the responses from a number of cells at low analyte concentrations, a substantial improvement in signal-to-noise ratio can be achieved and a corresponding reduction in the detection limit of the biosensor array is obtained.
A unique feature of the biosensor array of the present invention is that each of the individual cells and cell populations in the array may be encoded for maintaining cell type identity and location where randomly mixed populations of cells are employed. Cells may be encoded prior to disposing them in the microwells or, alternatively, following placement in the microwells. The invention provides for either encoding randomly mixed individual cells and cell populations with a fluorophoric or chromophoric dye compound or, alternatively, using self-encoded cells which are either naturally fluorescing or genetically engineered to fluoresce. Although cell populations may be randomly mixed together, this innovative feature provides for the identity and location of each cell type to be determined via a characteristic optical response signature when the cell array is either illuminated by excitation light energy or, alternatively, subjected to biological stimuli.
In one embodiment, cells or cell populations may be self-encoded by selecting cell populations, such as green fluorescent protein mutants, which exhibit either chemiluminescence, bioluminescence, or whose optical response to biological stimuli yield a unique detectable fluorescence signal. Other cell populations may be employed where cells within a population yield a unique temporal optical response to stimuli. Either naturally occurring of genetically engineered cell lines may be utilized.
In various alternative embodiments, cells may be encoded with dye compounds which are attached to cells, taken up by cells or provided in the local cell environment. Examples of useful encoding dyes include fluorophores, chromophores, stains or a dye compounds. For example, conventional cell fluorophore probes such as fluoresceins, rhodamines, naphthalimides, phycobiliproteins, nitrobenzoxadiazole may be utilized. A particularly useful reference for selecting appropriate encoding dyes is R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (6th ed.), Molecular Probes Inc.(Eugene, Oreg., 1996) which is herein incorporated by this reference.
When dye compounds are employed for encoding, cell populations within the biosensor array may be readily decoded by exciting the array with excitation light and indexing cells types within randomly dispersed populations by their response to excitation. In one embodiment, a single fluorophoric or chromophoric material or dye is used for encoding the cells. In an alternative embodiment, two or more encoding materials or dyes may be used to encode cell populations and the optical response intensity ratios for the dyes, produced by exposure to excitation light energy, are employed to encode and identify members of the cell population with the array. In an alternative embodiment, cells may be decoded by excitation light when exposed to a common analyte. In another embodiment, encoded cells may be decoded by their response to a generic cell activator using either a pH or Ca+2 indicator.
The innovative cell encoding feature of the present invention overcomes certain limitations of prior art devices by eliminating the need for mechanically scanning the array, mechanically indexing the location of cells, and mechanically positioning the array for measurements of individual cells within the array. The invention thus provides for rapid, simultaneous measurements of all cells and cell populations within the array without the need to mechanically scan the array to acquire a series of sequential measurements for each cell. Thus monitoring and measuring the responses of all cells in the array occurs simultaneously without a prolonged delay between the first cell measurement and last cell measurement. The ability to measure all cell responses simultaneously thus provides for the capability to monitor both short term cell response and long term cell response. This innovative feature thus enables the monitoring of rapid biologically significant cell processes and cell responses on a short time scale. In addition, the ability to simultaneously measure cell responses over a short time scale enables the measurement of individual cell and cell population response rates to changes in the biosensor array environment. This feature thus provides for additional discriminating response information which is useful for detecting biological or chemical analytes.
The biosensor array of the present invention can employ either naturally occurring cells and cell populations or genetically engineered cell lines. Virtually any cell type and size can be accommodated by matching the cell size to individual optical fiber optic core diameters and etching conditions. In one embodiment, NIH 3T3 mouse fibroblast cells were employed. In alternative embodiments, other cells types such as e. coli bacteria, staphylococcus bacteria, myoblast precursors to skeletal muscle cells, neutrophil white blood cells, lymphocyte white blood cells, erythroblast red blood cells, osteoblast bone cells, chondrocyte cartilage cells, basophil white blood cells, eosinophil white blood cells, adipocyte fat cells, invertebrate neurons (Helix aspera), mammalian neurons, or adrenomedullary cells, may be utilized as well. Any cell type or mixtures of cell population types may also be employed providing the microwell can accommodate the individual cell size.
The optical responses of individual cells and cell populations to chemical or biological stimuli are typically interrogated and detected by coupling individual cells with appropriate indicators which may be either fluorophores, chromophores, stains or a dye compounds. For example, conventional cell fluorophore probes such as fluoresceins, rhodamines, naphthalimides, phycobiliproteins, nitrobenzoxadiazole may be utilized. Alternatively, permeant or impermeant cell membrane potential indicators, ion indicators, reactive oxygen indicators and pH indicators may be employed. A particularly useful reference for selecting appropriate indicators is R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (6th ed.), Molecular Probes Inc.(Eugene, Oreg., 1996) which is herein incorporated by this reference. Any suitable indicator or combinations of indicators may be utilized provided the indicator does not compromise cell response. In a variety of alternative embodiments, indicators may be either incorporated directly into the cell, for example by attachment to the cell membrane, by absorption or injection into the cell cytoplasm, or added to the cell external environment, such as a fluid contained within the microwells. In an alternative embodiment, indicators may be attached to the surface of the microwells.
In summary, the biosensor array and sensing method of the present invention offers many distinct advantages in overcoming the limitations of prior art devices. The sensor arrays are easily fabricated from commercially available optical imaging fibers to yield a cost effective, high density, precisely formed, biosensor array without requiring any sophisticated machining or forming process. Since optical imaging fibers and fiber optic arrays are available in a wide variety of core diameters, most cell types and sizes may be accommodated in by the device and method of the present invention. In addition, cells can be readily dispersed into the microwell array in random fashion with no need for physical indexing or scanning to locate individual cells or cell populations due to the innovative cell encoding technique. Sensing methods and sensing systems which employ the biosensor and sensor array of the present invention avoids many of the limitations in manipulating cells encountered with prior art devices. Once cells are placed within the microwells of the array, conventional imaging systems and methods which employ an imaging camera and conventional optics, can monitor the response of thousands of cells simultaneously, eliminating requirements for mechanical scanning mechanisms. Analysis of measurement data is further facilitated by implementing commercially available imaging software to process images of the biosensor array using pattern recognition techniques combined with neural network and other statistical methods.
The biosensor array and sensing method of the present invention may be employed for a number of useful analytical applications where individual cells, which are chemically or biologically stimulated by the presence of a biological or chemical material in the local cell environment, will respond to their environment by producing an optically detectable response either due to the presence of an appropriate indicator or due to the characteristic optical response of particular cell types which exhibit either natural or genetically-engineered chemiluminescence or bioluminescence. The biosensor array and method of the present invention thus capitalizes on the ability of living cells to respond to biologically significant compounds. Since the selectivity of living cells for such compounds has considerable value and utility in drug screening and analysis of complex biological fluids, the biosensor of the present invention offers distinct advantages to high throughput screening of combinatorial libraries where hundreds of thousands of candidate compounds must be evaluated.